Naunyn-Schmiedeberg’s Arch Pharmacol (2000) 362 : 382–391 DOI 10.1007/s002100000315
R E V I E W A RT I C L E
Karl-Norbert Klotz
Adenosine receptors and their ligands
Published online: 21 September 2000 © Springer-Verlag 2000
Abstract The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors distinguished as A1, A2A, A2B and A3 receptors. Their presence on basically every cell makes them an interesting target for the pharmacological intervention in many pathophysiological situations. A large number of ligands have been synthesized over the last two decades and provide agonists and antagonists that are more or less selective for the known receptor subtypes. In addition, many radioligands are available in tritiated or radioiodinated form. The comparative pharmacological characterization of all four human adenosine receptor subtypes revealed that some of the compounds thought to be selective from data in other species have unexpected potencies at human receptors. As a result, compounds that exhibit high affinity to only one subtype are an exception. Although the selection of ligands is immense, it is less than satisfying for most subtypes of adenosine receptors. Key words Adenosine · Adenosine receptors · Agonist · Antagonist · Selectivity · High affinity · A1 · A2A · A2B · A3 · Ligand · Radioligand · Human
Introduction Adenosine is a well-known building block for many biologically relevant molecules like ATP, NAD+ or nucleic acids. The regulatory effects of adenosine are equally important and its action on the heart has been discovered already 70 years ago (Drury and Szent-Györgyi 1929). Later it was shown that adenosine mediates an increase of cAMP levels in the brain (Sattin and Rall 1970). After the discovery of an inhibition of adenylyl cyclase as a second K.-N. Klotz (✉) Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany e-mail:
[email protected], Fax: +49-931-2013539
signaling pathway, two subtypes of G protein-coupled receptors, A1 and A2, were differentiated on the basis of effector coupling and pharmacological profiles (Londos et al. 1980; van Calker et al. 1978, 1979). The identification of an increasing number of physiological functions mediated by adenosine suggested that adenosine receptors might be interesting targets for drug treatment of many different diseases (Daly 1982). This potential was significantly increased with the discovery of more receptor subtypes during the last decade. Although postulated for a number of years, only cloning confirmed the existence of additional subtypes (Pierce et al. 1992; Salvatore et al. 1993; Zhou et al. 1992). Currently, the P1 subfamily of purine receptors counts four subtypes distinguished as A1, A2A, A2B and A3 (Fredholm et al. 1994). The discovery of new adenosine receptor subtypes opened up new avenues for potential drug treatment of a variety of conditions such as asthma, neurodegenerative disorders, psychosis and anxiety, chronic inflammatory diseases and many other pathophysiological states that are believed to be associated with changes of adenosine levels (Jacobson et al. 1995; Müller and Stein 1996; Feoktistov et al. 1998; Poulsen and Quinn 1998). Selective and potent agonists and antagonists at the human receptor subtypes are needed for such therapeutic intervention. In particular, for A1 receptors many powerful ligands were developed during the 1980s. However, some of the wellknown pharmacological tools thought to be selective for A1 and A2 receptors were later recognized to be non-selective due to their unexpected affinity at A2B and A3 receptors. In addition, several ligands turned out to be less selective for human A1 and A2A receptor subtypes compared to other species typically used in previous studies. Thus, not only arose a need for novel compounds selective for the ‘new’ receptor subtypes, also some of the ligands for the ‘old’ receptors require improvement. It should be pointed out that marked species differences were observed for agonists as well as for antagonists (Ferkany et al. 1986; Ji et al. 1994; Klotz et al. 1991; Linden 1994; Stone et al. 1988), resulting in conflicting statements about selectivity of adenosine receptor ligands. In this review
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the consideration of affinity and selectivity is based on the human receptor subtypes unless specifically stated differently. This overview attempts to summarize the current availability of selective and potent ligands at human adenosine receptor subtypes.
Functional characteristics of adenosine receptors Adenosine receptors are of functional significance in many organ systems (for review see Ralevic and Burnstock 1998 and parallel review in this issue by Fredholm et al. 2000). Both A1 and A2A receptors play important roles in the cardiovascular system and the CNS. In the heart, adenosine causes negative chronotropic, dromotropic and inotropic effects which are mediated via the inhibitory A1 subtype. A2A adenosine receptors on endothelial and smooth muscle cells are responsible for adenosine-induced vasodilation. These two receptor subtypes are also involved in the regulation of neuronal activity. Stimulation of neuronal A1 Fig. 1 Structure of adenosine. The positions of the adenosine molecule that are available for modification to change receptor affinity and selectivity are marked with arrows. For details see text
Fig. 2 Structure of prototypical adenosine receptor agonists
receptors, which are predominantly expressed in the brain cortex, hippocampus, cerebellum and spinal cord, results in a reduced neurotransmitter release and neuronal firing. The most prominent CNS location of A2A receptors is the striatum where an intricate functional interaction with dopamine receptor signaling occurs. As opposed to A1 and A2A receptors which where detected on the protein level, in many tissues the analysis of tissue distribution for A2B and A3 receptors does rely on the presence of mRNA. The A2B receptor has been identified in basically every cell type but appears to be present at higher expression levels in various parts of the intestine and the bladder. This subtype may also have important functions in the regulation of vascular tone and function of mast cells. Expression levels for A3 adenosine receptors are generally low and are highly species dependent. Consequently, only limited knowledge about their physiological role is currently available.
Agonists The endogenous agonist adenosine (Fig. 1) is of limited interest as a tool for the investigation of adenosine receptors due to its susceptibility to extensive metabolism by a host of enzymes (for review see Deussen 2000). In addition, it is formed in membrane preparations that are utilized for binding or functional experiments, requiring the presence of adenosine deaminase in order to eliminate the effects of endogenous adenosine. Adenosine is, however, the structural core of all agonists known to date. Three positions in the molecule may be modified to increase affinity to specific receptor subtypes without destroying the agonistic activity: the 5’-position of the ribose and the
384 Table 1 Binding affinity of agonists at human A1, A2A and A3 adenosine receptor subtypes (Ki-values in nM). For A2B receptors EC50-values (nM) for the agonist-mediated stimulation of adenylyl cyclase activity in a membrane preparation are shown (IAB-MECA N6-4amino-3-iodobenzyladenosine5’-N-methyluronamide, Cl-IBMECA 2-chloro-N6-3-iodobenzyladenosine-5’-N-methyluronamide; for further abbreviations see text)
Table 2 Comparison of affinity and selectivity of agonists at rat (r) and human (h) adenosine receptor subtypes (Ki-values in nM). Selectivity ratios are the lowest values for the A1, A2A and A3 selectivity of CCPA, CGS 21680 and IBMECA, respectively, calculated from the available data
A1 Unmodified ribose R-PIA S-PIA CPA CCPA
2.0 75 2.3 0.8
5’-Modified ribose NECA CGS 21680 HENECA PENECA PHPNECA AB-MECA IB-MECA IAB-MECA Cl-IB-MECA
14 290 60 560 2.7 1,500 3.7 8.5 120
A1 CCPA
r h
CGS 21680
r h
IB-MECA
r h
0.4 0.8 2,600 290 54 3.7
2- and N6-positions of the purine (Fig. 1). Any of these modifications render the agonists metabolically stable.
Substitution at the N 6-position The first known subtype-selective adenosine derivatives were modified at the N6-position and showed A1 selectivity (Daly 1982). The relevant compounds are PIA (N6-(2phenylisopropyl)adenosine), CHA (N6-cyclohexyladenosine) and CPA (N6-cyclopentyladenosine; Fig. 2). PIA was instrumental for the classification of A1 receptors which, in contrast to A2 receptors, exhibit a marked stereoselectivity for the R-isomer (Daly 1982). In a series of 1-deaza analogues of adenosines it turned out that 2-chloro substitution in addition to an N6-cyclopentyl increases A1 selectivity (Cristalli et al. 1988). The respective modification in adenosine led to the development of 2-chloro-N6cyclopentyladenosine (CCPA) as the most potent and selective A1 receptor ligand characterized in rat brain (Klotz et al. 1989; Lohse et al. 1988a). Although this holds true for the human receptors as well (>3000-fold A1-selective vs. A2A), one should keep in mind that the A1 selectivity compared to the A3 adenosine receptor is only about 50to 70-fold (Klotz et al. 1998; Table 1). Currently no N6substitution is known that would provide a better differentiation between the A1 and A3 adenosine receptors.
A2A
A3
860 7,800 790 2,300
16 45 43 42
20 27 6.4 620 3.1 3,600 2,500 470 2,100
6.2 67 2.4 6.2 0.42 22 1.2 0.64 11
A2B
Reference
11,200 21,700 18,600 18,800
Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1998
2,400 88,800 – >100,000 1,100 51,500 11,000 25,200 –
Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1999 Klotz et al. 1999 Klotz et al. 1999 Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1999
A2A
A3
Selectivity ratios Reference
3,900 2,300
– 42
A2A/A1 9800 A3/A1 50
Lohse et al. 1988 Klotz et al. 1998
15 27
– 67
A1/A2A 170 A3/A2A 2
Jarvis et al. 1989 Klotz et al. 1998
A1/A3 50 A1/A3 3
Gallo-Rodriguez et al. 1994 Klotz et al. 1998
56 2,500
1.1 1.2
5’-Modification and substitution at the N 6-position The oxidation of the 5’-carbon of the ribose, amidation of the resulting uronic acid and N-substitution of the amide is the only type of modification that is tolerated in the ribose moiety of adenosine. Removal of the 2’- and 3’-hydroxy groups completely abolishes agonistic activity of the A1 receptor agonist N6-cyclohexyladenosine (Lohse et al. 1988b). The prototypical adenosine derivative NECA (5’-N-ethylcarboxamidoadenosine; or, chemically correct, adenosine-5’-N-ethyluronamide; Fig. 2) was originally considered to be an A2-selective compound (Londos et al. 1980) but turned out to be a non-selective agonist with high affinity at other receptor subtypes as well (Bruns et al. 1986; Hutchinson et al. 1989; Klotz et al. 1998). Although the affinity at the A2B receptor is much lower than at the other subtypes, it is one of the agonists with the highest potency at this subtype so far known (Klotz et al. 1998). The introduction of N6-substituents into 5’-modified adenosine derivatives may change the receptor affinity and selectivity in a rather dramatic manner. The first agonists with high affinity for A3 adenosine receptors were such compounds bearing N6-benzyl substituents in addition to 5’-modification (Gallo-Rodriguez et al. 1994). The preferred 5’-modification is the 5’-N-methyluronamide which resulted in the highest A3 potency compared to other modifications in this position of adenosine (Fig. 2). Several compounds of a series of N6-benzyl-5’-N-methyl-
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carboxamidoadenosines („MECA’s“) exhibit nanomolar affinity for the rat A3 receptor. N6-3-Iodobenzyladenosine-5’-N-methyluronamide (IB-MECA; Ki-value 1.1 nM at the rat A3) is about 50-fold selective compared to the A1 and A2A receptor subtypes (Gallo-Rodriguez et al. 1994; Table 2). The affinity of IB-MECA for the human A3 receptor is similar to the rat data whereas the human A1 receptor binds IB-MECA with almost the same affinity as the A3 subtype (Klotz et al. 1998; Tables 1, 2). N64-Aminobenzyladenosine-5’-N-methyluronamide (ABMECA) is the most A3-selective N6-benzyl derivative of MECA with 70- and 170-fold selectivity compared to the A1 and A2A subtypes, respectively. In particular the compounds with Ki-values at A3 receptors in the low nanomolar range exhibit also high affinity at A1 receptors (Table 1). An interesting observation is that 2-chloro substitution of IB-MECA reduces both A1 and A3 affinity whereas the same modification of CPA results in an increase of the potency at A1-receptors with no change of A3 affinity (Table 1).
5’-Modification and substitution at the 2-position The first adenosine derivative with some A2(A) selectivity was 2-(phenylamino)adenosine (CV-1808; Bruns et al. 1986). Further evaluation of 2-substitution led to the development of the NECA derivative CGS 21680 (2-[p-(2carboxyethyl)phenethylamino]adenosine-5’-N-ethyluronamide; Fig. 2) as an A2(A)-selective agonist which was 140-fold selective vs. A1 adenosine receptors in a rat model (Hutchinson et al. 1989). However, data at human receptors show a tremendous species variation for the A1 receptor with an over tenfold higher affinity of this compound for the human subtype (Klotz et al. 1998). In addition, with a similar affinity of CGS21680 for A3 receptors it can no longer be considered to be an A2A-selective agonist. It is, however, still the ligand of choice to distinguish A2A- and A2B-mediated effects. Substitution of the 2-position of NECA with alkynyl chains results in a decrease of A1 affinity and a concomittant increase of the potency at A2A receptors (Cristalli et al. 1992). Of this series of 2-substituted compounds tested at the rat receptors 2-hexynyl-NECA (2-hexyn1-yl-adenosine-5’-N-ethyluronamide, HENECA; Fig. 2) exhibits a 36-fold A2A selectivity compared to the A1 subtype. This selectivity is less pronounced for the human adenosine receptor subtypes. It also turned out that HENECA possesses significant affinity at rat A3 receptors (Siddiqi et al. 1995). This observation led to the identification of highly potent and selective A3 receptor agonists (Klotz et al. 1999). Data on human receptors reveal that PHPNECA (2-(3-hydroxy-3-phenyl)propyn-1-yl-adenosine-5’-N-ethyluronamide) is one of the most potent A3 agonists with a Ki-value of 0.4 nM, whereas PENECA (2(2-phenyl)ethynyladenosine-5’-N-ethyluronamide) is the most selective agonist (>100-fold) currently available (Klotz et al. 1999). Similar to the N6-benzyl derivatives there might be a moderate increase in A3 selectivity with the 5’-
N-methyl- over the 5’-N-ethyluronamide (K.-N. Klotz, G. Cristalli, unpublished observation).
Partial agonists A number of strategies have been pursued to develop partial agonists for adenosine receptors. Smaller alkyl substituents in 8-position of the purine ring of CPA reduced the A1 affinity 30- to 100-fold with a concomitant loss of agonistic activity (Roelen et al. 1996). The A2A affinity was affected in a similar order of magnitude, thus, A1 selectivity of the compounds was preserved. Modifications in the ribose moiety of adenosine resulted in various A1-selective compounds with reduced agonistic activity compared to classical agonists like CPA or R-PIA. Removal of the 2’- or 3’-hydroxy group of such full agonists resulted in a reduction of both affinity and functional activity (van der Wenden et al. 1995). The 3’deoxy compounds are about 20- to 30-fold less potent than the parent structures and 10- to 20-fold more potent than the corresponding 2’-deoxy derivatives. Interestingly, removal of both hydroxy groups in the case of CHA does not further affect affinity compared to 2’-deoxyCHA; however, the resulting dideoxy compound is a pure antagonist (Klotz et al. 1988; Lohse et al. 1988b). Another ribose modification resulting in partial agonists at A1 adenosine receptors is the introduction of a 5’thioether function. 5’-Ethylthio-CPA binds with high affinity to the rat A1 subtype (Ki-value 45 nM) with 65% intrinsic activity compared to CPA, as determined by receptor-mediated stimulation of [35S]GTPγS binding (van der Wenden et al. 1998). The respective 5’-modification in combination with N6-benzyl substituents known to increase A3 affinity (see above) resulted in the first partial agonists with high affinity at human A3 adenosine receptors (van Tilburg et al. 1999).
Antagonists The CNS effects of the adenosine receptor antagonist caffeine were enjoyed by a vast majority of people on our planet long before the physiological effects of adenosine were discovered. Caffeine is indeed the „most widely consumed behaviorally active substance in the world“ (Fredholm et al. 1999). The methylxanthines constitute the prototypical group of antagonists and modifications to the molecule resulted in a huge selection of derivatives some of which show distinct subtype selectivity. More recently, other structures including triazoloquinazolines, triazolotriazines, dihydropyridines and adenine derivatives served as the basis for a variety of non-xanthine antagonists.
Xanthines Table 3 summarizes pharmacological data of xanthine derivatives with different characteristics whose structures are shown in Fig. 3. The naturally occurring compounds
386 Table 3 Binding affinity of antagonists at adenosine receptor subtypes. For structures of the compounds see Figs. 3, 4, 5. All data are taken from Klotz et al. (1998) for the human receptors unless a different species or source of data is indicated. For the A2B adenosine receptor functional data are shown for comparison. Effector A1
activation was measured as adenylyl cyclase activity in a membrane preparation (AC) or by determination of cAMP levels in intact cells (cAMP). All data are Ki-values (nM). For details see Klotz et al. 1998 (BS-DMPX 8-(3-bromostyryl)-3,7-dimethyl-1propargylxanthine; for further abbreviations see text)
A2A
A3
Binding Xanthines Theophylline DPCPX
6,800 3.9 0.3b 29 3,000c 2,500d 1,200e
XAC MRS 1595 BS-DMPX Non-xanthines CGS 15943 SCH 58261 ZM 241385 MRS 1220 MRS 1334 8-Br-9-ethyladenine
3.5 290g 540a 305h > 100,000i 280j
1,700 130 340b 1.0 2,000c 260d 8.2e
86,000 4,000 – 92 670c >100,000d –
4.2 0.6g 1.4a 52h >100,000i 52j
51 >10,000g 270a 0.7h 2.7i 28,000j
aLinden
fJi
bLohse
gOngini
et al. 1999 et al. 1987 (rat A1 and A2A receptor) cKim et al. 1999 dUnpublished data, Klotz and Müller eMüller et al. 1997 (rat A and A 1 2A receptor)
A2B AC
cAMP
Binding
40,000 1,000 – 140 – >100,000d –
6,700 18 – 1.1 – – –
7,900a 50a – 7a 34c – –
910 – – – – 840j
66 5,000g 50g – – –
16f – 31a – – –
and Jacobson 1999 et al. 1999 hKim et al. 1996 (rat A and A 1 2A receptor) iJiang et al. 1997 (rat A and A 1 2A receptor) jCamaioni et al. 1998
Fig. 3 Structure of xanthine antagonists at adenosine receptors
like theophylline generally have affinities in the micromolar range with the highest affinity at the A2A adenosine receptor (Table 3). This receptor subtype appears to be relevant for the behavioral activation caused by caffeine (Ledent et al. 1997; Svenningsson et al. 1997).
The most important site of the xanthine structure that allows for the variation of the pharmacological profile is the 8-position. Interestingly, substituents which have been shown to increase A1 potency of adenosine derivatives when introduced in the N6-position result in a similar ef-
387
fect in the 8-position of xanthine derivatives. Consequently, DPCPX (8-cyclopentyl-1,3-dipropylxanthine; or CPX which is used as an alternative acronym) was the prototypical A1-selective antagonist with a 1000-fold higher potency at the rat A1 subtype (Ki-value 0.3 nM) compared to the rat A2A receptor (Bruns et al. 1987; Lohse et al. 1987). However, at the human A1 receptor tritiated DPCPX exhibits a KD-value of only 4 nM and the selectivity vs. the A2A subtype drops to about 30-fold (Klotz et al. 1998). Moreover, in whole-cell studies DPCPX was shown to antagonize agonist-induced cAMP accumulation at the A2B receptor with a Ki-value of 18 nM (Klotz et al. 1998). In recent binding studies a Ki-value of 50 nM confirmed the relatively high affinity of DPCPX at this receptor subtype (Linden et al. 1999). The comparison of functional and binding data for antagonists at A2B adenosine receptors reveals an interesting discrepancy (Table 3). One would expect that different functional tests would yield the same potency as determined in binding assays. However, for A2B receptors a consistent difference is found between experiments determining adenylyl cyclase activity in membrane preparations compared to the measurement of cAMP levels in whole cell assays (Brackett and Daly 1994; Table 3). Currently there is no explanation for this unexpected behavior. Most high affinity xanthines like DPCPX or XAC (xanthine amine congener; 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine) bear propyl substituents in 1- or 1- and 3-position (Table 3). A notable exception is a series of 3-methyl-1-propargylxanthines which, in combination with a 7-methyl-8-styryl substitution, typically display A2A selectivity (Müller et al. 1997, 1998a; Sauer et al. 2000). Fig. 4 Structure of non-xanthine antagonists at adenosine receptors
Recently, new xanthine antagonists with Ki-values below 50 nM for the human A2B receptor were identified (Kim et al. 1999). One compound, MRS 1595 (8-[4-[(carboxymethyl)oxy]phenyl]-1,3-dipropylxanthine-N,N-(2,3dimethylmaleyl)hydrazide) with a Ki-value of 34 nM, is 100- and 60-fold selective compared to human A1 and A2A receptors, respectively. Interestingly, the affinity of this compound at the rat A1 and A2A subtypes is 10- to 30fold higher compared to the human receptor subtypes (Kim et al. 1999). Although originally denoted as xanthine insensitive, some compounds like XAC (Table 3) and related compounds (Kim et al. 1999) bind with nanomolar affinity to human A3 adenosine receptors.
Non-xanthines The first non-xanthine adenosine receptor antagonist identified was the triazoloquinazoline CGS 15943 (5-amino9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazoline; Williams et al. 1987). In Fig. 4 the structure of this compound is shown in comparison to similar heterocyclic compounds developed in an attempt to find subtype-selective antagonists. Binding data for non-xanthine antagonists are summarized in Table 3. CGS 15943 is a nonselective antagonist with Ki-values in the low nanomolar range for A1 and A2A receptors and an approximately tenfold lower potency at the A2B and A3 subtypes (Klotz et al. 1998). Modification of the molecule led to the development of an A3selective compound, MRS 1220 (9-chloro-2-(2-furyl)5-[(phenylacetyl)amino][1,2,4]triazolo[1,5-c]quinazoline), with a subnanomolar affinity (Kim et al. 1996). Another somewhat similar compound, ZM 241385 (4-[2-[7-amino2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]-
388
ethyl]phenol), was initially characterized as an A2A-selective antagonist (Poucher et al. 1995). However, [3H]ZM 241385 was recently successfully used as a radioligand at A2B adenosine receptors (Ji and Jacobson 1999). Structural elements of both CGS 15943 and ZM 241385 are found in another non-xanthine compound with the code SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine; Zocchi et al. 1996). The A2A selectivity of SCH 58261 initially found for the rat receptor was confirmed for the human receptor (Ongini et al. 1999). A direct comparison of these heterocyclic compounds shown in Fig. 4 reveals that modification of a basic structure results in compounds with quite distinct receptor selectivity (Kim et al. 1996; Ongini et al. 1999). Jacobson and coworkers discovered 1,4-dihydropyridine derivatives as a novel chemical class of adenosine
receptor antagonists and recently described such compounds with nanomolar affinity at the human A3 receptor (Jiang et al. 1997). Compound MRS 1334 (3-ethyl-5-(4nitrobenzyl)-2-methyl-4-phenylethynyl-6-phenyl-1,4-dihydropyridine-3,5-dicarboxylate) shown in Fig. 5 displays a Ki-value of 2.7 nM at the human A3 subtype, whereas a Ki-value of 3850 nM was reported for the rat receptor. It is interesting to note that the structural difference between MRS 1353 (3-ethyl-5-[4-[(2,2,2-trichloroethoxy)carbonyl]benzyl]-2-methyl-4-phenylethynyl-6-phenyl-1,4-dihydropyridine-3,5-dicarboxylate) and MRS 1334 results in an almost 2000-fold difference in the affinity for the human A3 receptor whereas at the rat receptor only an approximately twofold difference was observed (Fig. 5). The observation that an intact ribose moiety is essential for agonistic activity led to the obvious strategy to develop adenosine receptor antagonists by replacing the ribose of adenosine with smaller substituents (see e.g. Thompson et al. 1991). Generally, these ligands are of moderate affinity and very rarely do they exhibit a pronounced preference for a single receptor subtype. 8-Bromo-9-ethyladenine is one of the most potent antagonists (and also one of the smallest compounds) with a Ki-value of 52 nM at the A2A receptor (Camaioni et al. 1998). The non-xanthine antagonists listed in this chapter represent a limited selection. Many more compounds have been developed over the years, some resembling more or less the xanthine structure or the antagonists selected in Fig. 4. Compounds were included in this review based on the availability of data for the human receptor subtypes.
Agonist radioligands Fig. 5 Comparison of two dihydropyridine derivatives as nonxanthine antagonists with marked pharmacological differences at rat vs. human A3 adenosine receptors Table 4 Radioligands for adenosine receptor subtypes. The data are KD-values for radiolabeled compounds or Kivalues from competition experiments (nM) for human receptors unless a different species is indicated (MSX-2 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthine)
One of the first high affinity radioligands for adenosine receptors was [3H]R-PIA which is still in use as an A1-se-
A1
A2A
A2B
Agonists [3H]PIA [3H]NECA [3H]CCPA [3H]CGS 21680
2.0 14 0.6 –
– 20 – 32
125I-AB-MECA
3.4
Antagonists [3H]DPCPX [3H]ZM 241385 125I-ZM 241385 [3H]SCH 58261 [3H]MSX-2 125I-ABOPX [3H]MRE 3008F20
A3
References
– – – –
16 6.2 – –
25
–
1.5
8.5
–
–
0.6
Klotz et al. 1998 Klotz et al. 1998 Klotz et al. 1998 Unpublished data, Klotz and Hessling (95% confidence limits 20–50 nM) Olah et al. 1994 (rat A1 and A3 receptor, canine A2A receptor) Klotz et al. 1998
3.9 0.3 – – – – – –
– – – 0.7 2 8 – –
50 – 33 – – – 36 –
– – – – – – – 0.8
Klotz et al. 1998; Linden et al. 1999 Lohse et al. 1987 Ji and Jacobson 1999 Palmer et al. 1995 (rat receptor) Dionisotti et al. 1997 Müller et al. 1998b Linden et al. 1999 Varani et. al. 2000
389
lective probe (Schwabe and Trost 1980). Subsequently, other N6-substituted adenosine derivatives became available as tritiated ligands including the agonist with the highest affinity and A1 selectivity, [3H]CCPA (Klotz et al. 1989). Originally introduced as a ligand for A2 receptors, [3H]NECA may now be considered to be a prototypical nonselective ligand. It labels A1, A2A and A3 receptors with similar affinity with a slight preference for the A3 subtype (Table 4). When CGS 21680 was introduced as an A2-selective agonist, it was also developed as a tritiated ligand (Jarvis et al. 1989). As discussed above, CGS 21680 is not an ideal tool for the characterization of A2A adenosine receptors, in particular if differentiation from A3 receptors is required. The tritiated compound displays a KD-value of 32 nM at the human A2A receptor and is, therefore, somewhat less potent than [3H]NECA (Table 4). The identification of A2A receptors in tissues with low receptor density is still a challenge. For the characterization of A3 adenosine receptors two agonistic radioligands are available none of which is selective for the human receptor subtype. As mentioned above, with a KD-value of 6 nM the nonselective [3H]NECA is a reasonable probe for A3 receptors if no other subtypes are present. The second radiolabeled agonist for A3 receptors is 125I-AB-MECA, allowing for the detection of receptors expressed at low levels due to the high specific radioactivity of radioiodinated compounds (Olah et al. 1994). However, this ligand shows also significant affinity at A1 receptors (Klotz et al. 1998; Olah et al. 1994; Table 4).
Antagonist radioligands Antagonist radioligands have the advantage over agonists that they will label all receptors present in a cell or membrane preparation independent of their coupling to a G protein and are, therefore, the preferred radioligands for the determination of receptor density. A common problem with the antagonists available for adenosine receptors is their low water-solubility and, thus, tendency for nonspecific binding. The only antagonist radioligand for the A1 adenosine receptor is [3H]DPCPX (or [3H]CPX). Although this xanthine derivative displays lower affinity at the human than the rat receptor (see above), it is still a useful tool for the characterization of A1 receptors and their discrimination from other subtypes. For A2A adenosine receptors a number of radiolabeled antagonists were developed over the years (Table 4). One of the most selective compounds, SCH 58261 (Ongini et al. 1999), shows high affinity binding at human A2A receptors with a KD-value of 2.3 nM for the tritiated ligand (Dionisotti et al. 1997). Another non-xanthine with high A2A affinity is ZM 241385 whose hydroxyphenyl structure allows for radioiodination (Palmer et al. 1995). This compound is also available as a tritiated ligand which binds with reasonable affinity (KD-value 34 nM) to the hu-
man A2B receptor (Ji and Jacobson 1999). Unfortunately, no comparative data are available about the A2A and A2B affinities for the tritiated vs. the iodinated compound. In addition to these non-xanthine radioligands, a 1-propargyl8-styrylxanthine was introduced as an A2A-selective tritiated compound (Müller et al. 1998b). Similarly to [3H]ZM 241385, the xanthine 125I-ABOPX (3-(4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine) exhibited a Ki-value of 36 nM at the A2B receptor and was recently characterized as an antagonist radioligand (Linden et al. 1999). Recently, the new non-xanthine antagonist MRE 3008F20 (5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine; Fig. 4) was introduced as a tritiated A3 antagonist. With a KD-value of 0.8 nM at the human A3 receptor and an over hundred- and thousandfold lower affinity at the A2A and A1 subtypes, respectively, it is a promising new tool for the characterization of A3 receptors (Varani et al. 2000). In addition to the development of an A3-selective agonist radioligand there is certainly room for improvement of currently available radioligands at all receptor subtypes.
Concluding remarks Over the last two decades an immense number of adenosine receptor ligands was synthesized, resulting in a selection of agonists and antagonists with different pharmacological characteristics. The modification of adenosine led to agonists with some selectivity for human A1 (CCPA) or A3 adenosine receptors (AB-MECA, PENECA); however, selective agonists for the A2 subtypes are still missing. On the antagonist side the number of selective compounds is limited as well. In addition to the prototypical xanthine derivatives several other structures are now available as antagonists at adenosine receptors. SCH 58261 exhibits a well-documented A2A selectivity whereas DPCPX lost its A1 selectivity to a significant potency at the A2B receptor. The dihydropyridine MRS 1334 is highly selective for the human A3 receptor when compared to rat A1 and A2A data. Overall, the availability of highly potent and selective adenosine receptor ligands is less satisfactory than it was a decade ago before the discovery and characterization of the A2B and A3 receptor subtypes. The therapeutic potential based on the distribution of the now known four adenosine receptor subtypes makes it a desirable goal to develop such agonists and antagonists. Acknowledgements Work in the author’s laboratory was supported in part by the BIOMED 2 program of the European Commission (EURCAR).
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References Brackett LE, Daly JW (1994) Functional characterization of the A2b adenosine receptor in NIH 3T3 fibroblasts. Biochem Pharmacol 47:801–814 Bruns RF, Lu GH, Pugsley TA (1986) Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29:331–346 Bruns RF, Fergus JH, Badger EW, Bristol JA, Santay LA, Hartman JD, Hays SJ, Huang CC (1987) Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes. Naunyn-Schmiedeberg’s Arch Pharmacol 335:59–63 Camaioni E, Costanzi S, Vittori S, Volpini R, Klotz K-N, Cristalli G (1998) New substituted 9-alkylpurines as adenosine receptor ligands. Bioorg Med Chem 6:523–533 Cristalli G, Franchetti P, Grifantini M, Vittori S, Klotz K-N, Lohse MJ (1988) Adenosine receptor agonists: synthesis and biological evaluation of 1-deaza analogues of adenosine. J Med Chem 31:1179–1183 Cristalli G, Eleuteri A, Vittori S, Volpini R, Lohse MJ, Klotz K-N (1992) 2-Alkynyl derivatives of adenosine and adenosine-5’-Nethyluronamides as selective agonists at A2 adenosine receptors. J Med Chem 35:2363–2368 Daly JW (1982) Adenosine receptors: targets for future drugs. J Med Chem 25:197–207 Deussen A (2000) Metabolic flux rates of adenosine in the heart. Naunyn-Schmiedeberg’s Arch Pharmacol DOI 10.1007/ s002100000318 Dionisotti S, Ongini E, Zocchi C, Kull B, Arslan G, Fredholm BB (1997) Characterization of human A2A adenosine receptors with the antagonist radioligand [3H]-SCH 58261. Br J Pharmacol 121:353–360 Drury AN, Szent-Györgyi A (1929) The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 68:213–237 Feoktistov I, Polosa R, Holgate ST, Biaggioni I (1998) Adenosine A2B receptors: a novel therapeutic target in asthma? Trends Pharmacol Sci 19:148–153 Ferkany JW, Valentine HL, Stone GA, Williams M (1986) Adenosine A1 receptors in mammalian brain: species differences in their interactions with agonists and antagonists. Drug Dev Res 9:85–93 Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden K, Jacobson KA, Leff P, Williams M (1994) VI: Nomenclature and classification of purinoceptors. Physiol Rev 46:143–156 Fredholm BB, Bättig K, Holmén J, Nehlig A, Zvartau EE (1999) Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev 51:83– 133 Fredholm BB, Arslan G, Kull B, Wassermann W, Schulte G (2000) Structure and function of adenosine receptors. NaunynSchmiedeberg’s Arch Pharmacol DOI 10.1007/s002100000313 Gallo-Rodriguez C, Ji X, Melman N, Siegman BD, Sanders LH, Orlina J, Pu Q-L, Olah ME, Galen PJM van, Stiles GL, Jacobson KA (1994) Structure-activity relationship of N6-benzyladenosine-5’-uronamides as A3-selective adenosine agonists. J Med Chem 37:636–646 Hutchinson AJ, Webb RL, Oei HH, Ghai GR, Zimmerman MB, Williams M (1989) CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity. J Pharmacol Exp Ther 251:47–55 Jacobson KA, Kim HO, Siddiqi SM, Olah ME, Stiles GL, Lubitz DKJE von (1995) A3-adenosine receptors: design of selective ligands and therapeutic prospects. Drugs Future 20:689–699 Jarvis MF, Schulz R, Hutchinson AJ, Do UH, Sills MA, Williams M (1989) [3H]CGS 21680, a selective A2 adenosine receptor agonist, directly labels A2 receptors in rat brain. J Pharmacol Exp Ther 251:888–893 Ji X-D, Jacobson KA (1999) Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors. Drug Des Discov 16:217–226
Ji X-D, Lubitz DKJE von, Olah ME, Stiles GL, Jacobson KA (1994) Species differences in ligand affinity at central A3adenosine receptors. Drug Dev Res 33:51–59 Jiang J, Rhee AM van, Chang L, Patchornik A, Ji X, Evans P, Melman N, Jacobson KA (1997) Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists. J Med Chem 40: 2596–2608 Kim Y-C, Ji X, Jacobson KA (1996) Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype. J Med Chem 39:4142–4148 Kim Y-C, Karton Y, Ji X, Melman N, Linden J, Jacobson KA (1999) Acyl-hydrazide derivatives of a xanthine carboxylic congener (XCC) as selective antagonists at human A2B adenosine receptors. Drug Dev Res 47:178–188 Klotz K-N, Lohse MJ, Schwabe U (1988) Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site. J Biol Chem 263:17522–17526 Klotz K-N, Lohse MJ, Schwabe U, Cristalli G, Vittori S, Grifantini M (1989) 2-Chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) – a high affinity agonist radioligand for A1 adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 340:679–683 Klotz K-N, Vogt H, Tawfik-Schlieper H (1991) Comparison of A1 adenosine receptors in brain from different species by radioligand binding and photoaffinity labelling. Naunyn-Schmiedeberg’s Arch Pharmacol 343:196–201 Klotz K-N, Hessling J, Hegler J, Owman C, Kull B, Fredholm BB, Lohse MJ (1998) Comparative pharmacology of human adenosine receptor subtypes – characterization of stably transfected receptors in CHO cells. Naunyn-Schmiedeberg’s Arch Pharmacol 357:1–9 Klotz K-N, Camaioni E, Volpini R, Kachler S, Vittori S, Cristalli G (1999) 2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 360:103–108 Ledent C, Vaugeois J-M, Schiffmann SN, Pedrazzini T, El Yacoubi M, Vanderhaeghen J-J, Costentin J, Heath JK, Vassart G, Parmentier M (1997) Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor. Nature 388:674–678 Linden J (1994) Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci 15:298–306 Linden J, Thai T, Figler H, Jin X, Robeva AS (1999) Characterization of human A2B adenosine receptors: radioligand binding, Western blotting, and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 56: 705–713 Lohse MJ, Klotz K-N, Lindenborn-Fotinos J, Reddington M, Schwabe U, Olsson RA (1987) 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) – a selective high affinity antagonist radioligand for A1 adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 336:204–210 Lohse MJ, Klotz K-N, Schwabe U, Cristalli G, Vittori S, Grifantini M (1988a) 2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 337:687–689 Lohse MJ, Klotz K-N, Diekmann E, Friedrich K, Schwabe U (1988b) 2’,3’-Dideoxy-N6-cyclohexyladenosine: an adenosine derivative with antagonist properties at adenosine receptors. Eur J Pharmacol 156:157–160 Londos C, Cooper DMF, Wolff J (1980) Subclasses of external adenosine receptors. Proc Natl Acad Sci USA 77:2551–2554 Müller CE, Stein B (1996) Adenosine receptor antagonists: structure and potential therapeutic applications. Curr Pharm Des 2:501–530 Müller CE, Geis U, Hipp J, Schobert U, Frobenius W, Pawlowski M, Suzuki F, Sandoval-Ramírez J (1997) Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists. J Med Chem 40:4396–4405
391 Müller CE, Sandoval-Ramírez J, Schobert U, Geis U, Frobenius W, Klotz K-N (1998a) 8-(Sulfostyryl)xanthines: water-soluble A2A-selective adenosine receptor antagonists. Bioorg Med Chem 6:707–719 Müller CE, Sauer R, Maurinsh Y, Huertas R, Fülle F, Klotz K-N, Nagel J, Hauber W (1998b) A2A-selective adenosine receptor antagonists: development of water-soluble prodrugs and a new tritiated radioligand. Drug Dev Res 45:190–197 Olah ME, Gallo-Rodriguez C, Jacobson KA, Stiles GL (1994) 125I4-Aminobenzyl-5’-N-methylcarboxamidoadenosine, a high affinity radioligand for the rat A3 adenosine receptor. Mol Pharmacol 45:978–982 Ongini E, Dionisotti S, Gessi S, Irenius E, Fredholm BB (1999) Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 359:7–10 Palmer TM, Poucher SM, Jacobson KA, Stiles GL (1995) 125I-4(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor. Mol Pharmacol 48: 970–974 Pierce KD, Furlong TJ, Selbie L-A, Shine J (1992) Molecular cloning and expression of an adenosine A2b receptor from human brain. Biochem Biophys Res Commun 187:86–93 Poucher SM, Keddie JR, Singh P, Stogall SM, Caulkett PWR, Jones G, Collis MG (1995) The in vitro pharmacology of ZM241385, a potent, non-xanthine A2a selective adenosine receptor antagonist. Br J Pharmacol 115:1096–1102 Poulsen S-A, Quinn RJ (1998) Adenosine receptors: new opportunities for future drugs. Bioorg Med Chem 6:619–641 Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492 Roelen H, Veldman N, Spek AL, Frijtag Drabbe Künzel J von, Mathôt RAA, IJzerman AP (1996) N6,C8-disubstituted adenosine derivatives as partial agonists for adenosine A1 receptors? J Med Chem 39:1463–1471 Salvatore CA, Jacobson MA, Taylor HE, Linden J, Johnson RG (1993) Molecular cloning and characterization of the human A3 adenosine receptor. Proc Natl Acad Sci USA 90:10365–10369 Sattin A, Rall TW (1970) The effect of adenosine and adenine nucleotides on the cyclic adenosine 3’,5’-phosphate content of guinea pig cerebral cortex slices. Mol Pharmacol 6:13–23 Sauer R, Maurinsh J, Reith U, Fülle F, Klotz K-N, Müller CE (2000) Water-soluble phosphate prodrugs of 1-propargyl-8styrylxanthine derivatives, A2A-selective adenosine receptor antagonists. J Med Chem 43:440–448 Schwabe U, Trost T (1980) Characterization of adenosine receptors in rat brain by (–)[3H]N6-phenylisopropyladenosine. Naunyn-Schmiedeberg’s Arch Pharmacol 313:179–187 Siddiqi SM, Jacobson KA, Esker JL, Olah ME, Ji X, Melman N, Tiwari KN, Secrist JA, Schneller SW, Cristalli G, Stiles GL, Johnson CR, IJzerman AP (1995) Search for new purine- and ribose-modified adenosine analogues as selective agonists and antagonists at adenosine receptors. J Med Chem 7:1174–1188
Stone GA, Jarvis MF, Sills MA, Weeks B, Snowhill EW, Williams M (1988) Species differences in high-affinity adenosine A2 binding sites in striatal membranes from mammalian brain. Drug Dev Res 15:31–46 Svenningsson P, Nomikos GG, Ongini E, Fredholm BB (1997) Antagonism of adenosine A2A receptors underlies the behavioral activating effect of caffeine and is associated with reduced expression of messenger RNA for NGFI-A and NGFI-B in caudate-putamen and nucleus accumbens. Neuroscience 79: 753–764 Thompson RD, Secuda S, Daly JW, Olsson RA (1991) N6,9-Disubstituted adenosines: potent, selective antagonists at the A1 adenosine receptor. J Med Chem 34:2877–2882 Van Calker D, Müller M, Hamprecht B (1978) Adenosine inhibits the accumulation of cyclic AMP in cultured brain cells. Nature 276:839–841 Van Calker D, Müller M, Hamprecht B (1979) Adenosine regulates, via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. J Neurochem 33:999–1005 Van der Wenden EM, Frijtag D von, Mathot RA, Danhof M, IJzerman AP, Soudijn W (1995) Ribose-modified adenosine analogues as potential partial agonists for the adenosine receptor. J Med Chem 38:4000–4006 Van der Wenden EM, Carnielli M, Roelen HC, Lorenzen A, Frijtag D von, Ijzerman AP (1998) 5’-Substituted adenosine analogs as new high-affinity partial agonists for the adenosine A1 receptor. J Med Chem 41:102–108 Van Tilburg EW, Frijtag Drabbe Künzel J von, Groote M de, Vollinga RC, Lorenzen A, IJzerman AP (1999) N6,5’-Disubstituted adenosine derivatives as partial agonists for the human adenosine A3 receptor. J Med Chem 42:1393–1400 Varani K, Merighi S, Gessi S, Klotz K-N, Leung E, Baraldi PG, Cacciari B, Romagnoli R, Spalluto G, Borea PA (2000) [3H]MRE 3008-F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A3 adenosine receptors. Mol Pharmacol 57:968–975 Williams M, Francis J, Ghai G, Braunwalder A, Psychoyos S, Stone GA, Cash WD (1987) Biochemical characterization of the triazoloquinazoline, CGS 15943, a novel, non-xanthine adenosine antagonist. J Pharmacol Exp Ther 241:415–420 Zhou Q-Y, Li C, Olah ME, Johnson RA, Stiles GL, Civelli O (1992) Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proc Natl Acad Sci USA 89:7432–7436 Zocchi C, Ongini E, Conti A, Monopoli A, Negretti A, Baraldi PG, Dionisotti S (1996) The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2a adenosine receptor antagonist. J Pharmacol Exp Ther 276:398–404
